Terminal Splice Variants Research Articles

Cystic Fibrosis Transmembrane Conductance Regulator Attaches Tumor Suppressor PTEN to the Membrane and Promotes Anti Pseudomonas aeruginosa Immunity

The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl-/- mice, which lack the NH2-amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport tothemembrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P.aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy.

Isolating and characterizing three alternatively spliced mu opioid receptor variants: mMOR-1A, mMOR-1O, and mMOR-1P

Extensive alternative pre-mRNA splicing of the mu opioid receptor gene, OPRM1, has demonstrated an array of splice variants in mice, rats and humans. Three classes of splice variants have been identified: full-length seven transmembrane (TM) domain variants with C-terminal splicing, truncated 6TM variants and single TM variants. The current studies isolates and characterizes an additional three full-length C-terminal splice variants generated from the mouse OPRM1 gene: mMOR-1A, mMOR-1O, and mMOR-1P. Using RT-qPCR, we demonstrated differential expression of these variants' mRNAs among selected brain regions, supporting region-specific alternative splicing. When expressed in Chinese Hamster Ovary cells, all the variants displayed high mu binding affinity and selectivity with subtle differences in the affinities toward some agonists. [³⁵S]γGTP binding assays revealed marked differences in agonist-induced G protein activation in both potency and efficacy among the variants. Together with the previous studies of mu agonist-induced phosphorylation and internalization in several carboxyl terminal splice variants, the current studies further suggest the existence of biased signaling of various agonists within each individual variant and/or among different variants.

Morphine Regulates Expression ofμ-Opioid Receptor MOR-1A, an Intron-Retention Carboxyl Terminal Splice Variant of theμ-Opioid Receptor (OPRM1) Gene via miR-103/miR-107

The μ-opioid receptor (MOR-1) gene OPRM1 undergoes extensive alternative splicing, generating an array of splice variants. Of these variants, MOR-1A, an intron-retention carboxyl terminal splice variant identical to MOR-1 except for the terminal intracellular tail encoded by exon 3b, is quite abundant and conserved from rodent to humans. Increasing evidence indicates that miroRNAs (miRNAs) regulate MOR-1 expression and that μ agonists such as morphine modulate miRNA expression. However, little is known about miRNA regulation of the OPRM1 splice variants. Using 3'-rapid amplification cDNA end and Northern blot analyses, we identified the complete 3'-untranslated region (3'-UTR) for both mouse and human MOR-1A and their conserved polyadenylation site, and defined the role the 3'-UTR in mRNA stability using a luciferase reporter assay. Computer models predicted a conserved miR-103/107 targeting site in the 3'-UTR of both mouse and human MOR-1A. The functional relevance of miR-103/107 in regulating expression of MOR-1A protein through the consensus miR-103/107 binding sites in the 3'-UTR was established by using mutagenesis and a miR-107 inhibitor in transfected human embryonic kidney 293 cells and Be(2)C cells that endogenously express human MOR-1A. Chronic morphine treatment significantly upregulated miR-103 and miR-107 levels, leading to downregulation of polyribosome-associated MOR-1A in both Be(2)C cells and the striatum of a morphine-tolerant mouse, providing a new perspective on understanding the roles of miRNAs and OPRM1 splice variants in modulating the complex actions of morphine in animals and humans.

Localization and Regulation of the N Terminal Splice Variant of PGC-1αin Adult Skeletal Muscle Fibers

The transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) regulates expression of genes for metabolism and muscle fiber type. Recently, a novel splice variant of PGC-1α (NT-PGC-1α, amino acids 1–270) was cloned and found to be expressed in muscle. Here we use Flag-tagged NT-PGC-1α to examine the subcellular localization and regulation of NT-PGC-1α in skeletal muscle fibers. Flag-NT-PGC-1α is located predominantly in the myoplasm. Nuclear NT-PGC-1α can be increased by activation of protein kinase A. Activation of p38 MAPK by muscle activity or of AMPK had no effect on the subcellular distribution of NT-PGC-1α. Inhibition of CRM1-mediated export only caused relatively slow nuclear accumulation of NT-PGC-1α, indicating that nuclear export of NT-PGC-1α may be mediated by both CRM1-dependent and -independent pathways. Together these results suggest that the regulation of NT-PGC-1α in muscle fibers may be very different from that of the full-length PGC-1α, which is exclusively nuclear.

Age and Visual Experience-dependent Expression of NMDAR1 Splice Variants in Rat Retina

The N-methyl-D-aspartate receptor (NMDAR) is a key molecule mediating brain plasticity related processes. Knowing that alternative splicing of the NMDAR1 (NR1) subunit offers molecular diversity to NMDAR, controls the forward trafficking of the NR1 protein and is important for placing NMDA receptors at synapses, we investigated herein the postnatal developmental expression and the influence of visual deprivation on NR1 subunit splice variants in rat retina. Real-time PCR was performed using oligonucleotide primers specific for N- terminal (NR1a, NR1b) and C-terminal splice variants (NR1-1, NR1-2, NR1-3, NR1-4). The developmental profiles of mRNA expression levels of all NR1 isoforms peaked at the end of the third week. Dark rearing led to reductions in both N- and C-terminal NR1 variants in several developmental ages and a significant interaction between age and visual experience was observed for NR1a, NR1-2 and NR1-4 expression. Our results have demonstrated a developmental and visual experience-dependent regulation of NR1 splicing in rat retina.

Long C terminal splice variant CaV2.2 identified in presynaptic membrane by mass spectrometric analysis

CaV2.2 voltage-gated calcium ­channels play a key role in the gating of transmitter release at presynaptic terminals. Recently we used mass spectrometry (MS) to analyze the protein complex associated with CaV2.2 in purified presynaptic terminal membranes. A number of known and new CaV2.2-associated proteins were identified, but not the channel itself. Here we set out to explore this anomaly. As previously, we used antibody Ab571 to capture the channel from purified synaptosome membrane lysate. We prepared a brain membrane lysate enriched for presynaptic active zones using standard methods to fractionate purified synaptosomes. These were osmotically lysed to generate a fraction enriched in presynaptic surface membranes. The lysate was solubilized in modified RIPA buffer and was passed over anti-CaV2.2 antibody covalently bonded to immunoprecipitation beads. Captured complexes on the beads were then stripped of weakly-bound proteins by exposure to high salt to enrich the channel fraction. Proteins remaining bound to the sample were recovered in high concentration urea and the sample was subjected to standard enzyme digestion and MS analysis. We identified 12 distinct CaV2.2 peptides, but no other ion channel peptides, in the lysate-exposed bead sample but no other ion channel peptides were recovered. Interestingly one of the channel peptides was derived from the alternatively spliced, long-C terminal region. Hence, confidence in identification of CaV2.2 was beyond reasonable doubt. The identification of the long-splice CaV2.2 provides compelling evidence that this variant is targeted to the presynaptic terminal, as we and others have suggested.

Long C terminal splice variant Cav2.2 identified in presynaptic membrane by mass spectrometric analysis

Long C terminal splice variant Cav2.2 identified in presynaptic membrane by mass spectrometric analysis

Immunohistochemical labeling of the mu opioid receptor carboxy terminal splice variant mMOR-1B4 in the mouse central nervous system

The mu opioid receptor gene Oprm is alternatively spliced into many variants, providing for the multiplicity of mu opioid receptor subtypes. One of the mouse variants, mMOR-1B4, is unique in that it displays high affinity towards a wide range of mu opioid receptor antagonists, but poor affinity towards most classical mu opioid agonists. The present study examined the immunohistochemical distribution of the mMOR-1B4 variant in mouse brain and spinal cord. mMOR-1B4-like immunoreactivity (mMOR-1B4-LI) was enriched in many regions of the brain, spinal cord and in the dorsal root ganglia. Some of the structures showing prominent mMOR-1B4-LI include the olfactory bulb, cerebral cortex, bed nucleus of stria terminalis, hippocampus, habenular nucleus, amygdala, thalamus, hypothalamus, medium eminence, substantia nigra, ventral tegmental area, oculomotor nucleus, red nucleus, raphe nuclei, periaqueductal gray, locus coeruleus, trigeminal nucleus, reticular formation, area postrema and Purkinje cell layer and deep nuclei of cerebellum. mMOR-1B4-LI was present in afferent neurons of the dorsal root ganglia and their projecting fibers into the superficial laminae of the spinal dorsal horn. Some motor neurons in the anterior horn of the spinal cord also were immunopositive. The overall distribution of mMOR-1B4-LI in the central nervous system is distinguishable from previously characterized variants such as MOR-1-LI, MOR-1C-LI and exon-11-LI. These studies provide evidence for the region- and neuron-specific processing of the Oprm gene and support the possibility of functional differences among the variants.

Long splice variant N type calcium channels are clustered at presynaptic transmitter release sites without modular adaptor proteins

The presynaptic N type Ca channel (CaV2.2) is associated with the transmitter release site apparatus and plays a critical role in the gating of transmitter release. It has been suggested that a distinct CaV2.2 long C terminal splice variant is targeted to the nerve terminal and is anchored at the release face by calcium/calmodulin-dependent serine protein kinase (CASK) and Munc-18-interacting protein (MINT), two modular adaptor proteins. We used the isolated chick ciliary ganglion calyx terminal together with two new antibodies (L4569, L4570) selective for CaV2.2 long C terminal splice variant to test these hypotheses. CaV2.2 long C terminal splice variant was present at the presynaptic transmitter release sites, as identified by Rab3a-interacting molecule (RIM) co-staining and quantitative immunocytochemistry. CASK was also present at the terminal both in conjunction with, and independent of its binding partner, MINT. Immunoprecipitation of CaV2.2 long C terminal splice variant from brain lysate coprecipitated CASK, confirming that these two proteins can form a complex. However, CASK was not colocalized either with CaV2.2 long C terminal splice variant or the transmitter release site marker RIM at the calyx terminal release face. Neither was MINT colocalized with CaV2.2 long C terminal splice variant. Our results show that native CaV2.2 long C terminal splice variant is targeted to the transmitter release sites at an intact presynaptic terminal. However, the lack of enrichment of CASK at the release site combined with the failure of this protein or its partner MINT to colocalize with CaV2.2 argues against the idea that these modular adaptor proteins anchor CaV2.2 at presynaptic nerve terminals.

Characterization of novel splice variants of the PAC 1 receptor in human neuroblastoma cells: Consequences for signaling by VIP and PACAP

Expression of VPAC and PAC 1 receptor isoforms was determined in six neuroblastoma cell lines as well as in human embryonic and adult brain using reverse transcriptase PCR and quantitative PCR. PAC 1 receptor splice variants missing a 21 amino acid sequence in the amino terminal domain were found to be the major receptor variants in the neuroblastoma cell lines and also were highly expressed in embryonic brain compared to adult brain. In four of the neuroblastoma cell lines, VIP and PACAP stimulated cyclic AMP production with different potencies and levels of maximal stimulation. High potency and greatest maximal stimulation of cyclic AMP for each peptide were recorded in SH-SY5Y cells, indicating the presence of high affinity VIP and PACAP receptors. Further characterization of specific VPAC and PAC 1 receptor isoforms was carried out in the SH-SY5Y cell line, where along with known PAC 1 receptor splice variants and the VPAC 2 receptor, a number of novel PAC 1 receptor splice variants were identified. The comparatively low level expression of the VPAC 2 receptor along with the poor responsiveness of SH-SY5Y cells to the VPAC 2 receptor-specific agonist Ro 25-1553 indicated that this receptor did not contribute significantly to the observed VIP responses. When the individual PAC 1 receptor isoforms were expressed in COS 7 cells, the ability of VIP to activate cyclic AMP production was increased more than 50-fold at the majority of the PAC 1 receptor variants lacking the 21 amino acid amino terminal domain sequence compared to those with the complete domain. Smaller changes were seen in the potency of PACAP-38. Similar trends were seen with inositol phosphate responses, where in each case agonist potencies were lower than for cyclic AMP production. The results of this study show that the combination of different amino terminal and intracellular loop 3 splicing variants in the PAC 1 receptor dictates the ability of agonists, particularly VIP, to activate signaling pathways. VIP has considerably greater potency at most PAC 1 receptors with the short amino terminal domain, and these therefore may mediate physiological effects of both VIP and PACAP. Furthermore, there may be a phenotypic switch in the expression of different PAC 1 receptor amino terminal splice variants between embryonic and mature nervous system, indicating that regulation of this event may have an important role in VIP/PACAP function, particularly in the developing nervous system.

Differential distribution in rat brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1-like immunoreactivity: evidence for region-specific processing.

The present study examined immunohistochemically the regional distribution of the mu opioid receptor splice variant MOR-1C by using a rabbit antisera generated against the C-terminal peptide sequences and compared it with MOR-1. Overall, the distribution of MOR-1C-like immunoreactivity (-LI) differed from MOR-1-LI. Both MOR-1C-LI and MOR-1-LI were prominent in a few central nervous system regions, including the lateral parabrachial nucleus, the periaqueductal gray, and laminae I-II of the spinal trigeminal nuclei and the spinal cord. In the striatum, hippocampal formation, presubiculum and parasubiculum, amygdaloid nuclei, thalamic nuclei, locus coeruleus, and nucleus ambiguous MOR-1-LI predominated, whereas MOR-1C-LI was absent or sparse. Conversely, MOR-1C-LI exceeded MOR-1-LI in the lateral septum, the deep laminae of the spinal cord, and most hypothalamic nuclei such as the median eminence, periventricular, suprachiasmatic, supraoptic, arcuate, paraventricular, ventromedial, and dorsomedial nuclei. Double-labeling studies showed colocalization of the two receptors in neurons of the lateral septum, but not in the median eminence or in the arcuate nucleus, even though both MOR-1 isoforms were expressed. Because both MOR-1 and MOR-1C are derived from the same gene, these differences in regional distribution represent region-specific mRNA processing. The regional distributions reported in this study involve the epitope seen by the combinations of exons 7, 8, and 9. However, if other MOR-1 variants containing exons 7, 8, and 9 exist, the antisera would not distinguish between them and MOR-1C.

Peripheral metabolic actions of leptin.

The ob gene product, leptin, is produced predominantly in adipose tissue. In ob/ob mice, a mutation in the ob gene prevents normal leptin production and results in a phenotype characterized by obesity and diabetes (Zhang et al. 1994). Daily injections of recombinant leptin to ob/ob mice inhibit food intake and reduce body weight and fat mass (Campfield et al. 1995; Halaas et al. 1995; Pelleymounter et al. 1995). This treatment also normalizes the blood glucose concentration and reduces the plasma insulin concentration of ob/ob mice (Pelleymounter et al. 1995). It was possible that the changes in glycaemia and insulin sensitivity in ob/ob mice given leptin might be a simple consequence of the antiobesity action. However, in pair-feeding studies, infusion of leptin not only resulted in a greater reduction in body fat than in the pair-fed mice, but the reduction in the plasma insulin concentration was much greater than that produced by an equivalent reduction in food intake (Levin et al. 1996). These findings suggested to us that leptin might have direct effects on a number of peripheral tissues associated with energy metabolism, including the endocrine pancreas and skeletal muscle. The leptin receptor (OB-R) is the product of the diabetes (db) gene (Chen et al. 1996; Lee et al. 1996) and was initially isolated as a complementary DNA (cDNA) from the choroid plexus cDNA library, encoding an 894 amino acid cell surface receptor which includes a hydrophobic signal sequence (Tartaglia et al. 1995). Structurally, OB-R is related to the class I cytokine receptor family, which includes gp 130, the common signal transducing chain for interleukin-6, leukaemia inhibitory factor receptor a-chain and the granulocyte colony-stimulatory factor. However, the interleukin-6, leukaemia inhibitory factor and granulocyte colony-stimulatory factor receptors all contain sequence motifs in the cytoplasmic domain that are required for interaction with janus kinase (JAK) and signal transduction and activation of transcription (STAT), whilst the OB-R from the choroid plexus lacks such a sequence. Further investigation showed that there are multiple isoforms of the OB-R, and a long form of the receptor (OB-Rb) was identified in the hypothalamus. It contained a long cytoplasmic domain of 302 amino acids, including consensus docking sites for JAK and STAT (Chen et al. 1996; Lee etal. 1996). The different isoforms of OB-Ra and OB-Rb are generated by alternative splicing, and additional short carboxyl terminal splice variants (OB-Rc, OB-Rd) and a soluble isoform lacking a transmembrane domain (OB-Re) have been identified also. The importance of the OB-Rb isofomi in the action of leptin was demonstrated by the finding of a point mutation (G + T transversion) in dh/db mice which results in a new splice donor site and, hence, a premature termination of translation (Chen et al. 1996; Lee et al. 1996). This truncated OB-R (db) isoform lacks the sequence motif required for JAK and STAT interaction and so db/db mice do not respond to leptin.